Multinozzle Emitter for Improved Negative Mode Analysis of Reduced Native N-Glycans by Microflow Porous Graphitized Carbon Liquid Chromatography Mass Spectrometry.

Microflow porous graphitized carbon liquid chromatography (PGC-LC) combined with negative mode ionization mass spectrometry (MS) provides high resolution separation and identification of reduced native N-glycan structural isomers. However, insufficient spray quality and low ionization efficiency of N-glycans present challenges for negative mode electrospray. Here, we evaluated the performance of a recently developed multinozzle electrospray source (MnESI) and accompanying M3 emitter for microflow PGC-LC-MS analysis of N-glycans in negative mode. In comparison to a standard electrospray ionization source, the MnESI with an M3 emitter improves signal intensity, identification, quantification, and resolution of structural isomers to accommodate low-input samples.

Veneer Is a Webtool for Rapid, Standardized, and Transparent Interpretation, Annotation, and Reporting of Mammalian Cell Surface N-Glycocapture Data.

Currently, no consensus exists regarding criteria required to designate a protein within a proteomic data set as a cell surface protein. Most published proteomic studies rely on varied ontology annotations or computational predictions instead of experimental evidence when attributing protein localization. Consequently, standardized approaches for analyzing and reporting cell surface proteome data sets would increase confidence in localization claims and promote data use by other researchers. Recently, we developed Veneer, a web-based bioinformatic tool that analyzes results from cell surface N-glycocapture workflows─the most popular cell surface proteomics method used to date that generates experimental evidence of subcellular location. Veneer assigns protein localization based on defined experimental and bioinformatic evidence. In this study, we updated the criteria and process for assigning protein localization and added new functionality to Veneer. Results of Veneer analysis of 587 cell surface N-glycocapture data sets from 32 published studies demonstrate the importance of applying defined criteria when analyzing cell surface proteomics data sets and exemplify how Veneer can be used to assess experimental quality and facilitate data extraction for informing future biological studies and annotating public repositories.

Surfaceome mapping of primary human heart cells with CellSurfer uncovers cardiomyocyte surface protein LSMEM2 and proteome dynamics in failing hearts.

Cardiac cell surface proteins are drug targets and useful biomarkers for discriminating among cellular phenotypes and disease states. Here we developed an analytical platform, CellSurfer, that enables quantitative cell surface proteome (surfaceome) profiling of cells present in limited quantities, and we apply it to isolated primary human heart cells. We report experimental evidence of surface localization and extracellular domains for 1,144 N-glycoproteins, including cell-type-restricted and region-restricted glycoproteins. We identified a surface protein specific for healthy cardiomyocytes, LSMEM2, and validated an anti-LSMEM2 monoclonal antibody for flow cytometry and imaging. Surfaceome comparisons among pluripotent stem cell derivatives and their primary counterparts highlighted important differences with direct implications for drug screening and disease modeling. Finally, 20% of cell surface proteins, including LSMEM2, were differentially abundant between failing and non-failing cardiomyocytes. These results represent a rich resource to advance development of cell type and organ-specific targets for drug delivery, disease modeling, immunophenotyping and in vivo imaging.

Bottom-up proteomic analysis of human adult cardiac tissue and isolated cardiomyocytes.

The heart is composed of multiple cell types, each with a specific function. Cell-type-specific approaches are necessary for defining the intricate molecular mechanisms underlying cardiac development, homeostasis, and pathology. While single-cell RNA-seq studies are beginning to define the chamber-specific cellular composition of the heart, our views of the proteome are more limited because most proteomics studies have utilized homogenized human cardiac tissue. To promote future cell-type specific analyses of the human heart, we describe the first method for cardiomyocyte isolation from cryopreserved human cardiac tissue followed by flow cytometry for purity assessment. We also describe a facile method for preparing isolated cardiomyocytes and whole cardiac tissue homogenate for bottom-up proteomic analyses. Prior experience in dissociating cardiac tissue or proteomics is not required to execute these methods. We compare different sample preparation workflows and analysis methods to demonstrate how these can impact the depth of proteome coverage achieved. We expect this how-to guide will serve as a starting point for investigators interested in general and cell-type-specific views of the cardiac proteome.

The effects of maturation and aging on the rotator cuff tendon-to-bone interface.

Rotator cuff tendon injuries often occur at the tendon-to-bone interface (i.e., enthesis) area, with a high prevalence for the elderly population, but the underlying reason for this phenomenon is still unknown. The objective of this study is to identify the histological, molecular, and biomechanical alterations of the rotator cuff enthesis with maturation and aging in a mouse model. Four different age groups of mice (newborn, young, adult, and old) were studied. Striking variations of the entheses were observed between the newborn and other matured groups, with collagen content, proteoglycan deposition, collagen fiber dispersion was significantly higher in the newborn group. The compositional and histological features of young, adult, and old groups did not show significant differences, except having increased proteoglycan deposition and thinner collagen fibers at the insertion sites in the old group. Nanoindentation testing showed that the old group had a smaller compressive modulus at the insertion site when compared with other groups. However, tensile mechanical testing reported that the old group demonstrated a significantly higher failure stress when compared with the young and adult groups. The proteomics analysis detected dramatic differences in protein content between newborn and young groups but minor changes among young, adult, and old groups. These results demonstrated: (1) the significant alterations of the enthesis composition and structure occur from the newborn to the young time period; (2) the increased risk of rotator cuff tendon injuries in the elderly population is not solely because of old age alone in the rodent model.

Safety, tolerability, and immune-biomarker profiling for year-long sargramostim treatment of Parkinson's disease.

BACKGROUND: Neuroinflammation plays a pathogenic role in Parkinson's disease (PD). Immunotherapies that restore brain homeostasis can mitigate neurodegeneration by transforming T cell phenotypes. Sargramostim has gained considerable attention as an immune transformer through laboratory bench to bedside clinical studies. However, its therapeutic use has been offset by dose-dependent adverse events. Therefore, we performed a reduced drug dose regimen to evaluate safety and to uncover novel disease-linked biomarkers during 5 days/week sargramostim treatments for one year. METHODS: Five PD subjects were enrolled in a Phase 1b, unblinded, open-label study to assess safety and tolerability of 3 µg/kg/day sargramostim. Complete blood counts and chemistry profiles, physical examinations, adverse events (AEs), immune profiling, Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS) scores, T cell phenotypes/function, DNA methylation, and gene and protein patterns were evaluated. FINDINGS: Sargramostim administered at 3 µg/kg/day significantly reduced numbers and severity of AEs/subject/month compared to 6 µg/kg/day treatment. While MDS-UPDRS Part III score reductions were recorded, peripheral blood immunoregulatory phenotypes and function were elevated. Hypomethylation of upstream FOXP3 DNA elements was also increased. INTERPRETATION: Long-term sargramostim treatment at 3 µg/kg/day is well-tolerated and effective in restoring immune homeostasis. There were decreased numbers and severity of AEs and restored peripheral immune function coordinate with increased numbers and function of Treg. MDS-UPDRS Part III scores did not worsen. Larger patient numbers need be evaluated to assess conclusive drug efficacy (ClinicalTrials.gov NCT03790670). FUNDING: The research was supported by community funds to the University of Nebraska Foundation and federal research support from 5 R01NS034239-25.

Facile Preparation of Peptides for Mass Spectrometry Analysis in Bottom-Up Proteomics Workflows.

Mass spectrometry (MS) is routinely used to identify, characterize, and quantify biological molecules. For protein analysis, MS-based workflows can be broadly categorized as top-down or bottom-up, depending on whether the proteins are analyzed as intact molecules or first digested into peptides. This article outlines steps for preparing peptide samples for MS as part of a bottom-up proteomics workflow, providing versatile methods suitable for discovery and targeted analyses in qualitative and quantitative workflows. Resulting samples contain peptides of suitable size for analysis by MS instrumentation generally available to modern research laboratories, including MS coupled to either liquid chromatography (LC) or matrix-assisted laser desorption/ionization (MALDI) interfaces. This article incorporates recent developments in methodologies and consumables to facilitate sample preparation. The protocols are well-suited to users without prior experience in proteomics and include methods for universally applicable suspension trap processing and for alternate in-solution processing to accommodate a range of sample types. Cleanup, quantification, and fractionation procedures are also described. © 2021 The Authors. Basic Protocol: Preparation of high-complexity peptide samples for mass spectrometry analysis using S-Trap™ processing Alternate Protocol 1: Preparation of low- to moderate-complexity peptide samples for mass spectrometry analysis using in-solution processing Alternate Protocol 2: Detergent, polymer, and salt removal from peptide samples before mass spectrometry analysis using SP2 processing Support Protocol 1: Protein quantification using Pierce 660 nm assay Support Protocol 2: Peptide quantification using Pierce quantitative fluorometric peptide assay Support Protocol 3: High-pH fractionation of complex peptide samples.

A year-long extended release nanoformulated cabotegravir prodrug.

Long-acting cabotegravir (CAB) extends antiretroviral drug administration from daily to monthly. However, dosing volumes, injection site reactions and health-care oversight are obstacles towards a broad usage. The creation of poloxamer-coated hydrophobic and lipophilic CAB prodrugs with controlled hydrolysis and tissue penetrance can overcome these obstacles. To such ends, fatty acid ester CAB nanocrystal prodrugs with 14, 18 and 22 added carbon chains were encased in biocompatible surfactants named NMCAB, NM2CAB and NM3CAB and tested for drug release, activation, cytotoxicity, antiretroviral activities, pharmacokinetics and biodistribution. Pharmacokinetics studies, performed in mice and rhesus macaques, with the lead 18-carbon ester chain NM2CAB, showed plasma CAB levels above the protein-adjusted 90% inhibitory concentration for up to a year. NM2CAB, compared with NMCAB and NM3CAB, demonstrated a prolonged drug release, plasma circulation time and tissue drug concentrations after a single 45 mg per kg body weight intramuscular injection. These prodrug modifications could substantially improve CAB's effectiveness.

Synthesis and Characterization of Long-Acting Darunavir Prodrugs.

Antiretroviral therapy (ART) has improved the quality of life in patients infected with HIV-1. However, complete viral suppression within anatomical compartments remains unattainable. This is complicated by adverse side effects and poor adherence to lifelong therapy leading to the emergence of viral drug resistance. Thus, there is an immediate need for cellular and tissue-targeted long-acting (LA) ART formulations. Herein, we describe two LA prodrug formulations of darunavir (DRV), a potent antiretroviral protease inhibitor. Two classes of DRV prodrugs, M1DRV and M2DRV, were synthesized as lipophilic and hydrophobic prodrugs and stabilized into aqueous suspensions designated NM1DRV and NM2DRV. The formulations demonstrated enhanced intracellular prodrug levels with sustained drug retention and antiretroviral activities for 15 and 30 days compared to native DRV formulation in human monocyte-derived macrophages. Pharmacokinetics tests of NM1DRV and NM2DRV administered to mice demonstrated sustained drug levels in blood and tissues for 30 days. These data, taken together, support the idea that LA DRV with sustained antiretroviral responses through prodrug nanoformulations is achievable.

Synthesis and characterization of a long-acting emtricitabine prodrug nanoformulation.

PURPOSE: A palmitoylated prodrug of emtricitabine (FTC) was synthesized to extend the drug's half-life, antiretroviral activities and biodistribution. METHODS: A modified FTC prodrug (MFTC) was synthesized by palmitoyl chloride esterification. MFTC's chemical structure was evaluated by nuclear magnetic resonance. The created hydrophobic prodrug nanocrystals were encased into a poloxamer surfactant and the pharmacokinetics (PK), biodistribution and antiretroviral activities of the nanoformulation (NMFTC) were assessed. The conversion of MFTC to FTC triphosphates was evaluated. RESULTS: MFTC coated with poloxamer formed stable nanocrystals (NMFTC). NMFTC demonstrated an average particle size, polydispersity index and zeta potential of 350 nm, 0.24 and -20 mV, respectively. Drug encapsulation efficiency was 90%. NMFTC was readily taken up by human monocyte-derived macrophages yielding readily detected intracellular FTC triphosphates and an extended PK profile. CONCLUSION: NMFTC shows improved antiretroviral activities over native FTC. This is coordinate with its extended apparent half-life. The work represents an incremental advance in the development of a long-acting FTC formulation.

Creation of a long-acting rilpivirine prodrug nanoformulation.

Antiretroviral therapy requires lifelong daily dosing to attain viral suppression, restore immune function, and improve quality of life. As a treatment alternative, long-acting (LA) antiretrovirals can sustain therapeutic drug concentrations in blood for prolonged time periods. The success of recent clinical trials for LA parenteral cabotegravir and rilpivirine highlight the emergence of these new therapeutic options. Further optimization can improve dosing frequency, lower injection volumes, and facilitate drug-tissue distributions. To this end, we report the synthesis of a library of RPV prodrugs designed to sustain drug plasma concentrations and improved tissue biodistribution. The lead prodrug M3RPV was nanoformulated into the stable LA injectable NM3RPV. NM3RPV treatment led to RPV plasma concentrations above the protein-adjusted 90% inhibitory concentration for 25 weeks with substantial tissue depots after a single intramuscular injection in BALB/cJ mice. NM3RPV elicited 13- and 26-fold increases in the RPV apparent half-life and mean residence time compared to native drug formulation. Taken together, proof-of-concept is provided that nanoformulated RPV prodrugs can extend the apparent drug half-life and improve tissue biodistribution. These results warrant further development for human use.

Synthesis of a long acting nanoformulated emtricitabine ProTide.

While antiretroviral therapy (ART) has revolutionized treatment and prevention of human immunodeficiency virus type one (HIV-1) infection, regimen adherence, viral mutations, drug toxicities and access stigma and fatigue are treatment limitations. These have led to new opportunities for the development of long acting (LA) ART including implantable devices and chemical drug modifications. Herein, medicinal and formulation chemistry were used to develop LA prodrug nanoformulations of emtricitabine (FTC). A potent lipophilic FTC phosphoramidate prodrug (M2FTC) was synthesized then encapsulated into a poloxamer surfactant (NM2FTC). These modifications extended the biology, apparent drug half-life and antiretroviral activities of the formulations. NM2FTC demonstrated a >30-fold increase in macrophage and CD4+ T cell drug uptake with efficient conversion to triphosphates (FTC-TP). Intracellular FTC-TP protected macrophages against an HIV-1 challenge for 30 days. A single intramuscular injection of NM2FTC, at 45 mg/kg native drug equivalents, into Sprague Dawley rats resulted in sustained prodrug levels in blood, liver, spleen and lymph nodes and FTC-TP in lymph node and spleen cells at one month. In contrast, native FTC-TPs was present for one day. These results are an advance in the transformation of FTC into a LA agent.

A long acting nanoformulated lamivudine ProTide.

A long acting (LA) hydrophobic and lipophilic lamivudine (3TC) was created as a phosphoramidate pronucleotide (designated M23TC). M23TC improved intracellular delivery of active triphosphate metabolites and enhanced antiretroviral and pharmacokinetic (PK) profiles over the native drug. A single treatment of human monocyte derived macrophages (MDM) with nanoformulated M23TC (NM23TC) improved drug uptake, retention, intracellular 3TC triphosphates and antiretroviral activities in MDM and CD4+ T cells. PK tests of NM23TC administered to Sprague Dawley rats demonstrated sustained prodrug and drug triphosphate levels in blood and tissues for 30 days. The development of NM23TC remains a substantive step forward in producing LA slow effective release antiretrovirals for future clinical translation.

Proteomic profiling of HIV-infected T-cells by SWATH mass spectrometry.

Viral pathogenesis results from changes in host cells due to virus usurpation of the host cell and the innate cellular responses to thwart infection. We measured global changes in protein expression and localization in HIV-1 infected T-cells using subcellular fractionation and the Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) proteomic platform. Eight biological replicates were performed in two independent experimental series. In silico merging of both experiments identified 287 proteins with altered expression (p < .05) between control and infected cells- 172 in the cytoplasm, 84 in the membrane, and 31 in nuclei. 170 of the proteins are components of the NIH HIV interaction database. Multiple Reaction Monitoring and traditional immunoblotting validated the altered expression of several factors during infection. Numerous factors were found to affect HIV infection in gain- and loss-of-expression infection assays, including the intermediate filament vimentin which was found to be required for efficient infection.

Two methods for proteomic analysis of formalin-fixed, paraffin embedded tissue result in differential protein identification, data quality, and cost.

Formalin-fixed paraffin-embedded (FFPE) tissue is a rich source of clinically relevant material that can yield important translational biomarker discovery using proteomic analysis. Protocols for analyzing FFPE tissue by LC-MS/MS exist, but standardization of procedures and critical analysis of data quality is limited. This study compared and characterized data obtained from FFPE tissue using two methods: a urea in-solution digestion method (UISD) versus a commercially available Qproteome FFPE Tissue Kit method (Qkit). Each method was performed independently three times on serial sections of homogenous FFPE tissue to minimize pre-analytical variations and analyzed with three technical replicates by LC-MS/MS. Data were evaluated for reproducibility and physiochemical distribution, which highlighted differences in the ability of each method to identify proteins of different molecular weights and isoelectric points. Each method replicate resulted in a significant number of new protein identifications, and both methods identified significantly more proteins using three technical replicates as compared to only two. UISD was cheaper, required less time, and introduced significant protein modifications as compared to the Qkit method, which provided more precise and higher protein yields. These data highlight significant variability among method replicates and type of method used, despite minimizing pre-analytical variability. Utilization of only one method or too few replicates (both method and technical) may limit the subset of proteomic information obtained.